Pharmacological depletion of microglia alleviates neuronal and vascular damage in the diabetic CX3CR1-WT retina but not in CX3CR1-KO or hCX3CR1I249/M280-expressing retina

Abstract

Diabetic retinopathy, a microvascular disease characterized by irreparable vascular damage, neurodegeneration and neuroinflammation, is a leading complication of diabetes mellitus. There is no cure for DR, and medical interventions marginally slow the progression of disease. Microglia-mediated inflammation in the diabetic retina is regulated <i>via</i> CX3CR1-FKN signaling, where FKN serves as a calming signal for microglial activation in several neuroinflammatory models. Polymorphic variants of <i>CX3CR1</i>, <i>hCX3CR1^I249/M280</i> , found in 25% of the human population, result in a receptor with lower binding affinity for FKN. Furthermore, disrupted CX3CR1-FKN signaling in <i>CX3CR1</i>-KO and <i>FKN</i>-KO mice leads to exacerbated microglial activation, robust neuronal cell loss and substantial vascular damage in the diabetic retina. Thus, studies to characterize the effects of <i>hCX3CR1^I249/M280</i> -expression in microglia-mediated inflammation in the diseased retina are relevant to identify mechanisms by which microglia contribute to disease progression. Our results show that <i>hCX3CR1^I249/M280</i> mice are significantly more susceptible to microgliosis and production of <i>Cxcl10</i> and <i>TNFα</i> under acute inflammatory conditions. Inflammation is exacerbated under diabetic conditions and coincides with robust neuronal loss in comparison to <i>CX3CR1</i>-WT mice. Therefore, to further investigate the role of <i>hCX3CR1^I249/M280</i> -expression in microglial responses, we pharmacologically depleted microglia using PLX-5622, a CSF-1R antagonist. PLX-5622 treatment led to a robust (~70%) reduction in Iba1⁺ microglia in all non-diabetic and diabetic mice. CSF-1R antagonism in diabetic <i>CX3CR1</i>-WT prevented TUJ1⁺ axonal loss, angiogenesis and fibrinogen deposition. In contrast, PLX-5622 microglia depletion in <i>CX3CR1</i>-KO and <i>hCX3CR1^I249/M280</i> mice did not alleviate TUJ1⁺ axonal loss or angiogenesis. Interestingly, PLX-5622 treatment reduced fibrinogen deposition in <i>CX3CR1</i>-KO mice but not in <i>hCX3CR1^I249/M280</i> mice, suggesting that <i>hCX3CR1^I249/M280</i> expressing microglia influences vascular pathology differently compared to <i>CX3CR1</i>-KO microglia. Currently <i>CX3CR1</i>-KO mice are the most commonly used strain to investigate CX3CR1-FKN signaling effects on microglia-mediated inflammation and the results in this study indicate that <i>hCX3CR1^I249/M280</i> receptor variants may serve as a complementary model to study dysregulated CX3CR1-FKN signaling. In summary, the protective effects of microglia depletion is <i>CX3CR1</i>-dependent as microglia depletion in <i>CX3CR1</i>-KO and <i>hCX3CR1^I249/M280</i> mice did not alleviate retinal degeneration nor microglial morphological activation as observed in <i>CX3CR1</i>-WT mice.